Kruppel-type zinc finger (ZNF) loci comprise one of the largest of all human gene families. In mammals, the majority of ZNF genes are of a single subtype, encoding proteins in which DMA-binding zinc finger arrays are attached to a chromatin-interacting domain, called KRAB, that confers a potent transcriptional repressor activity. Although certain KRAB-ZNF loci are highly conserved, ongoing segmental duplication events have created largely unique gene sets in each mammalian lineage. More than one-third of the 404 human KRAB- ZNF loci are primate-specific, and even the most recent duplicate genes have diverged in ways that indicate a selection for novel proteins with distinct DNA recognition sites. This single gene family comprises one-fifth of all predicted human transcription factor loci, and available data suggest broad roles in regulating processes that are critical to human health. However, since regulatory targets and pathways are known for only a handful of KRAB-ZNF proteins the functions of most family members remain a matter of conjecture. We hypothesize that this dynamic gene family has played a significant role in shaping human health-related biology, including both deeply conserved and primate-specific traits. The proposed research program is designed to address this hypothesis through functional analysis of 25 KRAB-ZNF genes. As a primary focus we will analyze proteins encoded by 25 genes involved in the most recent primate-specific duplications with emphasis on paralogous proteins that are most amenable to experimental analysis. Specifically, we will (1) determine genes and pathways that are regulated by each ZNF protein by manipulating gene expression in human cells;(2) define genomic regions to which the ZNF proteins bind in human chromatin using chromatin immunoprecipitation techniques;and (3) identify and validate consensus DNA motifs defining favored recognition sites for each protein using combined bioinformatics and experimental approaches. This study will provide a first in-depth look at functions for this large family of human transcriptional repressors, permitting us to examine their roles in regulating in human immunity, reproduction, development, cancer susceptibility and other processes in which KRAB-ZNF genes have been implicated. The data we generate will provide new guidelines to predict the functions of additional family members and to assess the potential impact of the gain, loss, mutation and dsyregulation of KRAB-ZNF genes on human health.